Adenovirus is a vector of choice for performing gene therapy. See, Jolly, D., Cancer Gene Therapy, vol. 1, no. 1, 1994: pp51-64. The well-characterized molecular genetics of adenovirus render it an advantageous vector in this regard. Adenoviruses are nonenveloped icosohedral double-stranded DNA viruses with a linear genome of approximately 36 kilobase pairs. Each end of the viral genome has a short sequence known as the inverted terminal repeat (or ITR), which is required for viral replication. Portions of the viral genome can be readily substituted with DNA of foreign origin, and furthermore, recombinant adenoviruses are structurally stable.
The adenovirus replication cycle has two phases: and early phase, during which 4 transcription units E1, E2, E3, and E4 are expressed, and a late phase which occurs after the onset of viral DNA synthesis when late transcripts are expressed primarily from the major late promoter (MLP). The late messages encode most of the virus's structural proteins. The gene products of E1, E2 and E4 are responsible for transcriptional activation, cell transformation, viral DNA replication, as well as other viral functions, and are necessary for viral growth.
To date most adenoviral vectors are based on viruses mutated in E1, E3 or a site upstream of E4 which provide for sites for the insertion of foreign DNA. The majority of vectors are based on adenovirus mutants which lack the E1 region of the genome. By deleting this region, the virus is rendered replication incompetent while simultaneously allowing for the insertion of foreign genes.
There are numerous reports on the use of adenovirus for gene therapy. For example, Smith, et al., Nature Genetics, Vol. 5, pgs. 397-402 (1993) discloses the administration to mice of an adenoviral vector including a human Factor IX gene. Such administration resulted in efficient liver transduction and plasma levels of human Factor IX that would be therapeutic for hemophilia B patients. Human Factor IX levels, however, slowly declined to baseline by nine weeks after injection, and were not re-established by a second vector injection. Smith, et al., also found that neutralizing antibodies to adenovirus block successful repeat administration of the adenovirus.
Kozarsky, et al., J. Biol. Chem., Vol. 269, No. 18, pgs. 13695-13702 (May 6, 1994) discloses the infusion of an adenoviral vector including DNA encoding the LDL receptor to rabbits. Stable expression of the LDL receptor gene was found in the rabbits for 7 to 10 days, and diminished to undetectable levels within 3 weeks. The development of neutralizing antibodies to the adenovirus resulted in a second dose being completely ineffective.
Kass-Eisler, et al., Gene Therapy, Vol. 1, pgs. 395-402 (1994) suggest that a T-cell response contributes to, but is not solely responsible for, the limited duration of expression in adults from adenovirus vectors. The authors further show that cyclosporin A is not effective in blocking the humoral response to the vector.
Fang, et al., J. Cell. Biochem., Supplement 21A, C6-109, pg 363 (1995) disclose the attempted re-injection of an adenovirus vector in dogs that were treated with cyclosporin A, an immunosuppressive agent. Such attempted re-injection was unsuccessful.
Yang, et al., Proc. Nat. Acad. Sci., Vol. 91, pgs. 4407-4411 (May 1994) describe recombinant adenoviruses in which the E1a and E1b regions have been deleted. Such viruses also include a heterologous gene. When these adenoviruses are administered to an animal host, cells harboring the recombinant viral genome express the heterologous gene as desired; however, low level expression of viral genes also occurs.
As exemplified above, adenoviruses can be efficient in gene transfer into cells in vivo, and thus may be employed as delivery vehicles for introducing desired genes into eukaryotic cells. There are, however, several limitations to adenovirus gene transfer which are due in part to host responses directed at either the adenovirus vector particle, breakdown products of the vector particle, or the transduced cells. These host responses include non-specific responses and specific immune responses. The non-specific responses include inflammatory and non-inflammatory changes. An example of the latter is a change in host cell gene expression. Specific immune responses include various cellular responses and humoral antibody responses. Cellular responses include those mediated by T-helper lymphocytes, T-suppressor lymphocytes, cytotoxic T lymphocytes (CTL), and natural killer cells.
Despite the high efficiency of adenovirus vector mediated gene transfer, the transient nature of adenovirus vector mediated gene transfer has suggested that repeat administrations of adenovirus vectors may be necessary. Recent studies in cotton rats, however, have demonstrated that host immune responses directed towards adenoviral vectors correlate with decreased efficiency of gene transfer and expression after repeated administration. Yei et al., Gene Therapy, 1: 192-200 (1994). The E3 region encodes several immunoregulatory proteins, which are not required for viral replication: gp19K, 10.4K, 14.5K and 14.7, and one protein, 11.6K, that is required for lysis of infected cells, and release of infectious progeny. Additionally, the E3 region also contains open reading frames for two proteins, 12.5k and 6.7k, whose functions have yet to be identified.
While the E3 region is not essential for viral replication, it does play a key role in modulating the host immune or inflammatory responses to the virus. For instance, in the case of the immune response it is known that gp19K binds to MHC class 1 molecules in the endoplasmic reticulum, thus inhibiting its glycoslation and transport to the surface of the virally infected cells. Consequently, the infected cells are not recognized as foreign by cytotoxic lymphocytes. See, Burgert, B., et al., Proc. Natl. Acad. Sci USA 1987; vol. 8: 1356-60.
Because of the many functions of the E3 region, it would be desirable to have an adenoviral vector for gene therapy applications that would permit one to delete particular regions of E3, and substitute foreign DNA, depending on the intended application of the vector. For example, there are described deletions in the E3 region that result in the removal of 1.88 kb between the Xbal sites. See, Berkner, K. and Sharp, P., (1983) Nucleic Acids Res. Vol. 11, pages 6003-6020, and Haj-Ahmad, Y. and Graham, F. (1986) J. Virol. Vol. 57, pages 267-274. Further, there is described compositions and methods for constructing adenovirus having insertions or deletions in both the E1 and E3 regions. See also, Ginsberg, H. S. et al., Proc. Natl. Acad. Sci. USA 1989, vol. 86, pp. 3823-7.
Thus, while these vectors have mutations in the E3 region, or large parts of the region deleted, to date there does not exist a vector that allows one to remove select parts of the E3 region and substitute foreign DNA, such that the expression of the substituted foreign, or heterologous DNA, retains the expression profile of the gene(s) deleted.